African trypanosomes are parasites of great medical and veterinary importance and useful for the study of the organization of the mitochondrial genome, regulation of mitochondrial gene expression and mutant mitochondrial DNA alteration. I propose to complete the cloning of the remaining 20% of the maxicircle DNA of T. brucei. These sequences and previously cloned maxicircle sequence will be employed to examine the detailed sequence organization of the maxicircle. Many cloned minicircles will be examined to determine minicircle sequence organization and sequence relationships among minicircles. The transcriptional organization of the maxicircle will be established. Bloodstream and culture form trypanosome maxicircle will be compared in order to elucidate the mechanism of regulation of mitochondrial (maxicircle) gene expression. Dyskinetoplastic (Dk) mutants will be examined using cloned kDNA (maxi- and mini-circle) probes. The loss or retention of specific maxicircle sequences and the alteration of specific kDNA sequence will be established in these mutants. Transcription of kDNA sequences in DK mutants will also be examined.